Supercritical angle fluorescence biosensor for the detection of molecular interactions on cellulose-modified glass surfaces

Cellulose films have been proposed as a convenient substrate for producing flat and homogeneous surface coatings. Additionally, amino-labelled cellulose species, like aminopropyltrimethylsilylethercellulose (ATMSC), are excellent support matrices for covalent binding of biomolecules, with low probe density and prevention of non-specific adsorption of unbound analyte molecules. Due to ATMSC films fulfil important requirements as substrate for analyse techniques of surface-tethered proteins and nucleic acids, we consequently report a new preparation for DNA-functionalised surfaces. Single-stranded DNA molecules are covalently coupled to cellulose-coated glass cover slips to interact with complementary free Cy5-labelled oligonucleotides in solution. Hybridisation efficiencies at the new substrate and at standard surface coatings are determined by detection of the surface-generated fluorescence. In order to discriminate against the fluorescence from unbound oligonucleotides the detection volume was restricted to the surface by collecting supercritical angle fluorescence (SAF). Thus, it is demonstrated that cellulose films are utilised to investigate DNA-hybridisation reactions highly sensitive.     

Fluorescence fluctuation spectroscopy in subdiffraction focal volumes

We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. Our method applies fluorescence correlation spectroscopy and fluorescence intensity distribution analysis to extract molecular information about mobilities and fluorescence emission in solution. The combination of correlation analysis with that of photon intensity distributions reveals a fivefold squeezing of the detection volume over current diffraction-limited systems, which is in full agreement with the simultaneously demonstrated 25-fold reduction in (axial) focal transit time. Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations.     

Creating attoliter detection volume by microsphere photonic nanojet and fluorescence depletion

In this paper, a novel method is presented for creating a 3-dimensinal sub-diffraction effective observation volume based on microsphere photonic nanojet and fluorescence depletion effect. Using microsphere to achieve photonic nanojet focusing effect, the proposed method applies the radially polarized plane wave to excite fluorescence and the azimuthally polarized beam to obtain fluorescence depletion field. The effective detection volume of photonic nanojet can reach 0.002 μm³ (2aL). Compared with conventional confocal microscopy, this effective detection volume represents a reduction of almost 2 orders of magnitude. With simple configuration based on cost-effective microspheres, the proposed method is theoretically proved to be a potential tool for the fluorescence correlation spectroscopy (FCS) to have large analysis range and to investigate single molecule at high concentrations.     

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

The interaction between a free-base, anionic water-soluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a bi-exponential decay is obtained where a short rotational correlation time (phi (int) = 1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin J-aggregates is induced, which can be detected by time-resolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sample.     

Resolution of <Emphasis Type="BoldItalic">λ</Emphasis> /10 in fluorescence microscopy using fast single molecule photo-switching

We demonstrate nanoscale resolution in far-field optical microscopy based on photo-switching of molecules. By enabling, recording and disabling fluorescence from individual labels sequentially, the detection volume is reduced to the size of a single molecule and the diffraction limit is broken. Images of nanostructures milled into a coverslip and tagged by fluorescent proteins could be recorded at 50 nm resolution. Due to the fast and asynchronous image acquisition protocol used in these experiments, we were able to reduce acquisition times to ∼2.5 min, which is two orders of magnitude lower than in previous implementations. PACS 81.07.-b; 42.30.-d; 87.64.-t     

Fluorescence Correlation Spectroscopy (FCS): A Promising Tool for Biological Research

Abstract

Fluorescence correlation spectroscopy (FCS) is an important biophysical technique. FCS is currently being used in many areas of biology to solve several scientific problems. Its properties such as detection at the single molecular level, higher sensitivity, and use of lower sample volume make FCS a promising molecular diagnostic tool. The promising applications of FCS extend from DNA kinetics/dynamics studies to the comprehensive understanding of receptor–ligand interactions. In this article, we review various promising biological applications of FCS.     

Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform

We have developed a filterless multicolor detection unit for fluorescence correlation spectroscopy (FCS). This grating-based setup is continuously tunable for multicolor separation and is thus a powerful alternative to the classical cascade of dichroic mirrors and filters. Our tailored platform allows for accommodation of up to 15 detection channels covering the entire visible spectral range. As a proof of principle, we successfully demonstrate simultaneous FCS of four distinct fluorescent quantum dot species being mixed in aqueous solution. Grating-based detection allows for spectral high-resolution FCS in a stable and compact setup and is a feasible tool for quantitative investigation of complexbiomolecular dynamics on a single molecule level.     

Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy

We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al (5 x 10(-18) l) at a water-glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.     

Multiscale modeling of alloy solidification using a database approach

A two-scale model based on a database approach is presented to investigate alloy solidification. Appropriate assumptions are introduced to describe the behavior of macroscopic temperature, macroscopic concentration, liquid volume fraction and microstructure features. These assumptions lead to a macroscale model with two unknown functions: liquid volume fraction and microstructure features. These functions are computed using information from microscale solutions of selected problems. This work addresses the selection of sample problems relevant to the interested problem and the utilization of data from the microscale solution of the selected sample problems. A computationally efficient model, which is different from the microscale and macroscale models, is utilized to find relevant sample problems. In this work, the computationally efficient model is a sharp interface solidification model of a pure material. Similarities between the sample problems and the problem of interest are explored by assuming that the liquid volume fraction and microstructure features are functions of solution features extracted from the solution of the computationally efficient model. The solution features of the computationally efficient model are selected as the interface velocity and thermal gradient in the liquid at the time the sharp solid–liquid interface passes through. An analytical solution of the computationally efficient model is utilized to select sample problems relevant to solution features obtained at any location of the domain of the problem of interest. The microscale solution of selected sample problems is then utilized to evaluate the two unknown functions (liquid volume fraction and microstructure features) in the macroscale model. The temperature solution of the macroscale model is further used to improve the estimation of the liquid volume fraction and microstructure features. Interpolation is utilized in the feature space to greatly reduce the number of required sample problems. The efficiency of the proposed multiscale framework is demonstrated with numerical examples that consider a large number of crystals. A computationally intensive fully-resolved microscale analysis is also performed to evaluate the accuracy of the multiscale framework.     

Quantification of Low Concentrations of DNA Using Single Molecule Detection and Velocity Measurement in a Microchannel

We present a novel method for quantifying low concentrations of DNA based on single molecule detection (SMD) for molecular counting and flow measurements inside a microchannel. A custom confocal fluorescence spectroscopic system is implemented to detect fluorescent bursts emitted from stained DNA molecules. Measurements are made one molecule at a time as they flow through a femtoliter-sized laser focal probe. Durations of single molecule fluorescent bursts, which are found to be strongly related to the molecular transit times through the detection region, are statistically analyzed to determine the in situ flow speed and subsequently the sample volume flowing through the focal probe. Therefore, the absolute concentration of a DNA sample can be quantified based on the single molecule fluorescent counts from the DNA molecules and the associated probe volume for a measured time course. To validate this method for quantifying low concentrations of biomolecules, we tested samples of pBR322 DNA ranging from 1 pM to 10 fM (∼3 ng/ml to 30 pg/ml). Besides molecular quantification, we also demonstrate this method to be a precise and non-invasive way for flow profiling within a microchannel.     

饱和激发情况下的荧光关联谱测量

研究了激发光增强时其对荧光关联谱(FCS)测量的影响. 强激发光产生的饱和激发会改 变激光诱导荧光的空间分布函数,从而影响FCS的测量结果. 首先根据饱和吸收的物理模型,推导出强激发光情况下有效探测区域变化的定量公式,获得了FCS测量所得到的粒子数和扩散时间与饱和激发光强和激光光强的函数关系,并用于消除饱和吸收造成的系统误差.采用Monte-Carlo模拟方法和实验,对理论分析结果进行了验证. 这将有助于完善FCS分析方法的理论模型,为高激发光强度下的FCS探测提供依据. 关键词： 荧光关联谱 饱和激发 Monte-Carlo模拟     

Phase-referenced interferometer with subwavelength and subhertz sensitivity applied to the study of cell membrane dynamics

We report a highly sensitive means of measuring cellular dynamics with a novel interferometer that can measure motional phase changes. The system is based on a modified Michelson interferometer with a composite laser beam of 1550-nm low-coherence light and 775-nm CW light. The sample is prepared on a coverslip that is highly reflective at 775 nm. By referencing the heterodyne phase of the 1550-nm light reflected from the sample to that of the 775-nm light reflected from the coverslip, small motions in the sample are detected, and motional artifacts from vibrations in the interferometer are completely eliminated. We demonstrate that the system is sensitive to motions as small as 3.6 nm and velocities as small as 1 nm/s. Using the instrument, we study transient volume changes of a few (approximately three) cells in a monolayer immersed in weakly hypotonic and hypertonic solutions.     

Chiral molecules split light: Reflection and refraction in a chiral liquid

A light beam changes direction as it enters a liquid at an angle from another medium, such as air. Should the liquid contain molecules that lack mirror symmetry, then it has been predicted by Fresnel that the light beam will not only change direction, but will actually split into two separate beams with a small difference in the respective angles of refraction. Here we report the observation of this phenomenon. We also demonstrate that the angle of reflection does not equal the angle of incidence in a chiral medium. Unlike conventional optical rotation, which depends on the path-length through the sample, the reported reflection and refraction phenomena arise within a few wavelengths at the interface and thereby suggest a new approach to polarimetry that can be used in microfluidic volumes.     

Real-Time Detection of Polymerase Activity Using Supercritical Angle Fluorescence

We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP-Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.     

On the Resolution Capabilities and Limits of Fluorescence Lifetime Correlation Spectroscopy (FLCS) Measurements

Quantitative tests were performed in order to explore the practical limits of FLCS. We demonstrate that: a) FLCS yields precise and correct concentration values from as low as picomolar to micromolar concentrations; b) it is possible to separate four signal components in a single detector setup; c) diffusion times differing only 25% from each other can be resolved by separating a two component mixture based on the different fluorescence lifetimes of both components; d) most of the inherent technical limitations of conventional FCS are easily overcome by FLCS employing a single detector channel confocal detection scheme.     

A fluorescence spectroscopy study of traditional Chinese medicine Angelica

By measuring the fluorescence spectra of Chinese medicine (CM) Angelica water solutions with different concentrations from 0.025 to 2.5 mg/mL, results showed that the fluorescence intensity was proportional to the concentration. Through fluorescence spectra of Angelica solution under different pH values, results indicated coumarin compounds were the active ingredients of Angelica. We also observed fluorescence quenching of the Angelica solution in the presence of spherical silver nanoparticles with radius of 12 nm. Keeping a certain value for the volume of the silver nanoparticles, the fluorescence intensity at 402 nm was linearly proportional to the Angelica in the range of 1–3 mg/mL.     

Diffusivity in water and fluorescence properties of?organic nanoparticles produced in?flames

Rotational and translational diffusion properties in water of nano organic carbon (NOC) particles collected from premixed laminar ethylene/air flames have been investigated using both time resolved fluorescence polarisation anisotropy (TRFPA) and fluorescence correlation spectroscopy (FCS). Insight into the NOC sizes, structures and rigidity has been gained through diffusion properties exploiting their fluorescence in different spectral ranges. The TRFPA measurements revealed the presence of two classes of particles. The first composed of particles with a mean size of 1.5?nm which absorb and fluoresce in the UV, and a second class composed of slightly larger particles, about 2.2?nm, which absorb and fluoresce in the visible and were also detected by FCS. From FCS measurements particle concentration and fluorescence quantum yield have been evaluated.     

热解析富集-能量色散X射线荧光光谱法对溶液中汞的测定

搭建热解析富集装置对溶液中的汞富集后进行检测,可以提高X射线荧光测试的灵敏度.整个测试过程如下:样品中的汞在高温下发生热解析,经过滤膜时被选择性吸附,在使用光谱仪测试后,最终计算出样品中汞的含量.在热解析管路中加入白云石增加停留时间,可以降低汞的热解还原温度,在使用汞稳定化剂的条件下,只需加热到600℃就可以实现汞的解...     

A scanning laser induced fluorescence detection system for capillary electrophoresis microchip based on optical fiber

In this paper, a scanning laser induced fluorescence detection system for capillary electrophoresis microchip based on single-mode optical fiber has been established using a green diode pumped solid-state laser as excitation source. It includes laser induced fluorescence detection subsystem, capillary electrophoresis microchip, mechanical scanning platform, channel identification unit and fluorescent signal processing subsystem. Two V-shaped detecting probes composed of optical fiber for transmitting the excitation light and detecting the induced fluorescence were constructed. Parallel four-channel signal analysis of capillary electrophoresis was performed by this system using Rhodamine B sample solution. Furthermore, the separation and distinction of different concentrations of Rhodamine B sample were achieved with the constructed detection system. The lowest detected concentration is 1.0 × 10⁻⁵ mol/L for Rhodamine B sample. The results show that the constructed detection system possesses some advantages of compact structure, low cost, and better stability which could be used as reference to the design and optimization of laser induced fluorescence detection system for capillary electrophoresis microchip.     

A Statistical Analysis of Fluorescence Correlation Data

Fluorescence correlation spectroscopy (FCS) is a relatively recent technique in which the diffusion coefficient of fluorescently labeled molecules can be determined. The change in diffusion behavior when these molecules interact with others can also be used to study interactions in solution. A new statistical method is proposed to analyze FCS measurements that cannot be evaluated with a classical autocorrelation function, which is normally used to analyze FCS data. It applies to binding studies where one of the interacting particles has a much brighter fluorescence intensity with respect to the other, which causes high fluorescence bursts whenever these molecules are detected. This biases the autocorrelation function, making it in most cases impossible to use this function as a fitting equation. Here, a statistical approach is used to quantify the amount of fluorescence found in bursts, thereby enabling to perform binding studies in cases where the fluorescence per molecule of both interacting species differs greatly. The method is demonstrated on a system of known composition, making it a promising tool for future FCS measurements.